Production of nucleic acid molecules can be fairly simple or complex depending on factors such as the type of nucleic acid molecules to be produced. For example, historically, short single stranded nucleic acid molecules such as primers have been typically generated by chemical synthesis (see, e.g., U.S. Pat. No. 5,837,858, the disclosure of which is incorporated herein by reference). Further, longer nucleic acid molecules have typically been generated by polymerase chain reaction (PCR). One disadvantage of PCR is that generally template nucleic acid is required.
Many nucleic acid synthesis methods have limited capabilities for the generation of large de novo nucleic acid molecules. One aspect of the current disclosure is to address this limitation.
Furthermore, nucleic acid molecules that are used for gene synthesis are usually produced using expensive automated machines with limited throughput. For this reason, alternative approaches are being investigated, such as the use of microarrays as a source for nucleic acid molecules for gene synthesis. Microarrays can have hundreds of thousands of different nucleic acid molecules on a small surface and can be fabricated at very low cost.
However, approaches to nucleic acid molecule synthesis that are based on microarrays may suffer from drawbacks such as low amounts of nucleic acid molecules produced per spot. This may not be problematic for the use of hybridization assays, the application for which two-dimensional microarrays were initially developed. However, when two-dimensional microarrays are used for gene synthesis (i.e., nucleic acid molecules are fabricated on a planar surface) they may lack at least two important features. First, the quantity of nucleic acid molecules may not be large enough to assemble a fragment, such as a fragment that can be used for gene assembly. In certain instances only attomoles may be obtained. For this reason, the nucleic acid molecules generated typically need to be copied by PCR to reach quantities that are useful for fragment assembly. Moreover, the quantity of nucleic acid molecules may be further reduced by synthesis reagents, such as acids, that can act to degrade the nucleic acid molecules after synthesis.
Second, it is difficult to release synthesized nucleic acid molecules from microarrays individually, or in pools needed for the assembly of one fragment. Rather, the nucleic acid molecules are typically released together, often resulting in complex pools of thousands of different nucleic acid molecules that may not be amenable for gene synthesis without post-processing. Complicated processes like dial-out PCR or sequencing for identification and amplification of the desired nucleic acid molecules are thus often needed to make use of these pools.